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卵泡抑素诱导骨髓间充质干细胞分化为神经元样细胞
李伟伟,蕾*,,李鹏飞
辽宁医学院生物化学教研室,锦州 121001
[摘要] ?目的:研究卵泡抑素(follistatin)对骨髓间充质干细(BMSCs)向神经细胞分化的影响。方法:利用 Percoll 密度梯度离心法及贴壁筛选法分离培养和扩增BMSCs通过流式细胞术分析鉴定BMSCs的纯度,follistatin诱导第三代生长良好的MSCs向神经元转化,观察分化过程中细胞形态的变化;利用RT-PCR方法检测诱导前后BMSCs的神经元特异性烯醇化酶(NSE)和神经干细胞标记物巢蛋白(Nestin)的表达情况结果:流式分析获得了纯度较高的BMSCs,细胞表达CD29CD44CD106CD166,不表达CD34CD45诱导10 d后,细胞呈现双极、多极和锥形的典型神经元细胞形态,并且在mRNA水平证明诱导分化后的细胞与对照组比较NSENestin的表达增加(P < 0.05结论:结果表明follistatin可以在体外诱导BMSCs分化为神经样细胞。
[关键词]骨髓;间充质干细胞;卵泡抑素;分化;神经元样细胞
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Differentiation of mesenchymal stem cells into neuron-like cells induced by follistatin
Li WeiweiZhang LeiTian LiLi Pengfei
(Department of Biochemistry, Liaoning Medical College, Jinzhou 121000, China)
[Abstract] Objective: To study the effect of follistatin on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into neuron-like cells. Methods: The human BMSCs were isolated and purified by density gradient centrifugation, and cultured by plastic-adhering. Then the purity of BMSCs was analyzed by flow cytometry. The differentiation of the third passage of BMSCs into neuron-like cells was induced by follistatin. Also the cell morphology of BMSCs during the differentiation was observed. The expressions of nestin and neuron specific enolase (NSE) of BMSCs were detected by RT-PCR. The high purity of BMSCs was collected. Results: The BMSCs expressed CD29, CD44, CD106 and CD166, but not CD34 and CD45. The results showed that, after 10 days of the differentiation of BMSCs by follistatin, the majority of the BMSCs were transformed into neuron-like cells. There were several processes (axon-like or dendrite-like) from the cell body. The levels of the expressions of nestin and NSE mRNAs on follistatin-treated BMSCs were increased significantly, compared with those on BMSCs without follistatin treatment (P < 0.05). Conclusion: The present results indicate that follistatin can induce human BMSCs to differentiate into neuron-like cells.
[Keywords] bone marrow; mesenchymal stem cell; follistatin; differentiation; neuron-like cells
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基金项目:(如果是,注明基金名称和编号)
*通讯作者:张蕾,?电话:0416-4673253, Emailzhbud@hotmail.com
作者简介:李伟伟,硕士,电话:13614962217E-mail:woshifeng-531@163.com
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骨髓间充质干细胞(BMSCs)是骨髓中存在的一类非造血组织干细胞,具有高度增殖和自我更新能力,在一定的环境和刺激因子作用下可分化为间质组织细胞,如成肌细胞(包括心肌细胞)、骨细胞、软骨细胞、神经细胞及脂肪细胞等,且在体外培养的过程中始终保持其多向分化的潜能[1-3]。因为BMSCs具有多向分化潜能和自体利用的特性,而且还可以诱导免疫耐受[4],所以将其作为种子细胞用于组织工程已成为当前研究的热点。Ueno [5]1987年首次从猪和牛卵泡液中分离获得的单链糖蛋白,具有抑制内分泌中枢垂体释放促性腺激素(FSH)的作用,同时也是激活素(activin)特异结合蛋白,它的mRNA在多种组织中都有表达[6,7]Hamoshimato[8]1992年的实验表明,follistatin能够抑制activin的活性,促进神经母细胞瘤细胞IMR-32的轴突延伸。目前中枢神经系统疾病引起的神经系统功能障碍最佳的治疗方法仍是神经细胞移植,达到功能修复和重建。但是,神经细胞移植的组织来源一直是难以解决的问题。因此,深入研究定向诱导MSCs分化为神经细胞的方法将有可能为中枢神经系统疾病的治疗提供大量种子细胞,具有深远的意义。本实验采用follistatin诱导体外培养的BMSCs,探讨follistatinBMSCs的诱导分化作用。
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材料和方法
1 ?材料
? 健康自愿者8,抽取髂骨骨髓5 mlFollistatin 购自Peprotech AsiaUSA批号:1306CY12,溶于超纯水,超滤除菌,-20保存。L-DMEM GibcolUSA)、胎牛血清(GibcolUSA)、0.25 %胰酶(Sigma)、Percoll分离液(Pharmacia)、RT-PCR试剂盒(Progema)、单克隆荧光抗体(CoulterUSA)。PCR仪(Eppendorf,德国)、超净工作台(苏州净化,苏州)、倒置相差显微镜(Olympus)、细胞培养箱(SANYO)和流式细胞仪(Coulter)。
2 方法
2.1骨髓间充质干细胞(BMSCs)的分离、原代培养及传代 肝素抗凝收集的骨髓液5 mlPBS稀释(体积比为1:1),缓慢加到等体积的Percoll1.073 g/ ml)分离液上,1500 r/min离心20 min。收集单个核细胞层,PBS3次,然后用含10 %胎牛血清的L-DMEM重悬细胞,制成单细胞悬液[9]。调整细胞密度为5 ×106 /L,接种于25 cm2培养瓶,置于37 5 %CO295 %湿度孵箱常规培养。48 ~72 h后更换新鲜培养液,去除未贴壁细胞,以后每3 d更换一次培养液。待细胞生长达85%融合时,弃去原来的培养基,PBS冲洗两次,加入0. 25 %胰蛋白酶0.8 ml,室温消化1 min。镜下观察见大部分细胞突起缩回,间隙变大,细胞近圆形为度,轻轻敲打瓶底使细胞漂浮,加入含10%胎牛血清的L-DMEM终止消化,收集细胞于离心管中,1500 r/min离心5 min。弃上清,10 %胎牛血清的L-DMEM重悬细胞,按1?2比例传代。经过几次传代,BMSCs即可得到纯化。
2.2 BMSCs表面标志鉴定?取成功传代培养的第2代细胞,0.25%胰酶消化,制成单个核细胞悬液,血球计数仪计数,调整细胞密度为为1×106 /LPBS洗涤后分别与抗CD29CD44CD106CD166CD34CD45单克隆抗体室温避光反应20 minPBS洗涤后应用流式细胞仪检测。
2.3 ?BMSCs向神经细胞分化的诱导方法?取生长状况良好的传至第三代的BMSCs用于实验,以2×105/L密度接种于6孔细胞培养板,当细胞生长至60%融合时,弃去培养液,PBS洗两次。诱导组加入诱导培养液(L-DMEM+10%FBS+follistatin),按照follistatin的浓度,诱导组可分为0.3 nmol/L组、3 nmol/L组、空白对照组(不加任何诱导剂),倒置显微镜下记录细胞形态变化。诱导48 h后换用完全培养基,第10天时收取细胞。
2.4 神经细胞的鉴定?1×106 诱导前后的BMSCs常规提取总RNA采用NSENestin特异性引物扩增目的片断,β-actin作为内参照。NSE 上游引物5'-aactgcccctgtatcgcca-3';下游引物5'-ttttccgtgtagccagcct-3'Nestin上游引物5'-tccagaaactcaagcaccac-3';下游引物5'-ccaccgtatcttcccacct-3'β-actin 上游引物5'-gggaaatcgtgcgtgacat-3',下游引物5'-caggaggagcaatgatctt-3';逆转录反应为10μl体系。PCR反应为25 μl体系,30个循环,反应条件和操作均参照试剂和说明。cNDA反转录条件:10 μl体系,42逆转录30 minPCR条件:50 μl体系,预变性 95℃ 5 min;变性95 30 s;退火57 30 s;延伸72 45 s30个循环;最后延伸72 10 min
? 统计学处理:电泳图像经Bandscan电泳图像处理系统分析, 测出每个扩增产物条带的PCR DNA吸光度值, 以扩增产物β-actin的吸光度值为对照,其他扩增产物与β-actin 吸光度值的百分比(%)表示NSENestin mRNA的表达水平。采用SPSS13.0软件进行方差分析,数据以表示,P < 0.05说明差异有统计学意义。
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结果[表注释用中文,图注释用英文]
1 ?BMSCs的形态学和表面标志鉴定
???分离的BMSCs胞体小,呈圆形,培养24 h后开始贴壁生长,3 d后可见较多贴壁细胞呈单个或多个细胞的克隆;贴壁细胞呈长梭形,成纤维细胞样,形态均匀,呈极性排列,换液去除漂浮细胞。8 d后细胞可达到90%融合,用胰酶和EDTA消化传代后细胞贴壁速度加快,增殖能力更强,细胞呈极性排列,形态均一(Fig.1)。流式细胞仪检测的结果显示,BMSCs表面抗原CD44CD29CD166阳性率达到90%以上,不表达或低表达CD14CD45CD34等(Fig.2)。
2 诱导分化后细胞生长情况和形态变化?
? Follistatin诱导后48 h即可见少量细胞的胞体变圆,长梭形的两极逐渐变细变长。诱导后第3天可见较多的形态类似神经元的细胞出现,这些细胞间以长的突起相互连接。诱导7 d时出现较多数量的具有1~3个细长突起、胞体呈圆形或椭圆形的神经元样细胞(Fig.3A)。诱导10 d时细胞形态与诱导7 d时的形态类似(Fig.3B)。
3 RT-PCR检测诱导前后BMSCs的神经细胞特异性标志物的表达
? ?未诱导的细胞NestinmRNA有弱表达。Follistatin诱导10 d后,0.3 nmol/l3nmol/l浓度组细胞表达Nestin mRNA均增加(P < 0.05,且3 nmol/l浓度组较0.3 nmol/l浓度组更高(P < 0.051)。未诱导细胞表达极弱NSE mRNAfollistatin诱导10 d后,0.3 nmol/L3nmol/l浓度组细胞表达NSE mRNA均增加(P < 0.05,且3 nmol/L浓度组较0.3 nmol/L浓度组更高(P < 0.051)。
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A
B
A: The primary cultured BMSCs show fusiform shape, but the unadherent cells are round shape (×200); B: the cells have oval shape limpidnucleolus after wright statining (×400).
Fig. 1 Morphological characteristics of undifferentiated BMSCs

CD44
CD44
CD29
CD166

Fig. 2 Flow cytometric analysis of cultured BMSCs with CD45, CD34, CD44CD166 and CD29 antibodies (The results indicate that BMSCs express CD29, CD44 and CD166, but not CD34 and CD45)
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A
B
A: After seven days of follistatin induction, the cell bodies show round or oval shape (×200). B: After 10 days of follistatin induction, the number of cells has no change (×200).
Fig. 3 Morphological characteristics of differentiated BMSCs
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??? 1 诱导前后NestinNSEmRNA表达量(n = 8,x±s

?巢蛋白(Nestin)
NSE mRNA
未诱导组
0.3 nmol/L
3 nmol/L
0.029 ± 0.002
0.169 ± 0.007a
0.159 ± 0.008c
0.033 ± 0.0040
0.173 ± 0.010a
?0.190 ± 0.011c

aP < 0.05 vs 未诱导组;cP < 0.05 vs 0.3 nmol/L组。
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讨论
近年来,许多研究表明BMSC能够在体外诱导分化呈神经细胞,体外诱导BMSCs向神经细胞分化的试剂主要包括视黄酸(RA)、生长因子(单独或联合使用)、抗氧化剂、脱甲氧试剂、环磷腺苷试剂、中药单体黄芩[10-12]等。Sanchez -Ramos[13 ]证实,在小牛血清、表皮生长因子(EGF)、RA和脑源性神经营养因子(brain-derived neurotropic factorBDNF)诱导下,BMSCs可分化为神经元样细胞,分化后的细胞表达神经干细胞特异性标志物巢蛋白(nestin)、神经元特异性核蛋白(neuron specific nuclear proteinNeuN)和星形胶质细胞标志物胶质纤维酸性蛋白(glial fiber acidic proteinGFAP)。Hashimoto等通过体外培养证实follistatin抑制P19的生长,且能够在体外诱导IMR-32细胞分化为神经细胞,但是没有检测到特异性的神经细胞的标志物表达[8]
Nestin属于第类中间丝蛋白,仅在胚胎早期神经上皮表达,一旦神经前体细胞朝向终末方向分化成神经元和胶质细胞Nestin便停止表达[14]。本实验PT-PCR结果显示未诱导的BMSCs和诱导后细胞均表达NestinNSE,说明诱导后的一部分细胞仍是未发生终末分化的神经前体细胞。
神经元的分化受许多细胞外信号分子的影响,这些分子通过细胞膜受体介导级联反应或核受体介导作用影响早期中枢神经系统的发育[15-16]Lidia][17认为smad3在运动神经元前体区的缺失是脊椎运动神经元正常发育的先决条件;Shoji-Kasai[18]Ageta[19]研究都证实了Activin能够促进神经细胞的发育。Activin是中胚层的诱导因子,在多种生物的生长发育中都具有重要的意义,而follistatin能够与activin的受体相结合,抑制其活性[20]Hashimoto(1992)通过比较RAfollistatin对体外培养IMR-32细胞的诱导分化作用,证实follistatin能够促进IMR-32细胞轴突延长,胞体变成圆形或椭圆形,其诱导作用比RA更加明显。本实验利用密度梯度离心法体外分离BMSCs,经follistatin诱导3 d后,即发现细胞胞体突起回缩,变圆,折光性增强。镜下可见细胞出现较长的树突和轴突,形成单极、双极和多极神经元形态。诱导后7 d ,仍有90 %以上的细胞存活,结果证实follistatin能够诱导神经分化,与Hashimoto[8]的结论相一致。
综上所述,activinfollistatin都可以诱导神经细胞的分化,而两者的作用又相互拮抗,所以有待于进一步研究其促进神经分化的作用机制,更好了解神经发育过程中的信号通路以及基因的调控。
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参考文献
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(收稿日期2009-04-10)

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